We believe that tumor cell lysis by cytotoxic T lymphocytes (CTL) is physiologically distinct from that mediated by antibody plus complement (Ab\plus c'). The two forms of lysis can be distinguished by monitoring the fate of the tumor cell nucleus. We will extend these observations to other forms of immune lysis as a preliminary step towards elucidating the relative importance of various forms of immune protection in different anatomical sites in the whole animal. Our observations have led us to propose that CTL-initiated lysis is an autolytic event carried out by the target after receiving a signal from the CTL. Using both murine and human effector systems, we will attempt to elucidate early events in the target cell after CTL attack and formulate a more precise mechanism of CTL-induced lysis. In addition, we will continue our studies on a model of a tumor escape mechanism on a model with a murine tumor that undergoes cyclical changes in CTL sensitivity despite a constant expression of H-2 antigen. A major effort will be a definitive characterization of the role of the CTL K+i/K+ gradient in the lytic process. Earlier experiments from our laboratory demonstrated that with mixed cultures, replacing extracellular Na+ with K+ selectively blocked a postbinding event in CTL lysis. Recently, we have found that mixing prelabeled (86Rb+) CTL with targets results in accelerated 86Rb+ release from the CTL. This accelerated 86Rb+ release is antigen specific and has the physical and physiological requirements that link it to the lethal hit stage of lysis. Over the course of the next year we will make quantitative measurements of the membrane potential of CTL (voltage sensitive dyes) after treatment with various agents and correlate the effect of membrane depolorization with lytic efficiency of the CTL. In addition, we will attempt to correlate the accelerated 86Rb+ release with other potential signals related to the lytic sequence within the CTL. Of particular interest here will be to determine if Ca+2 uptake is related to K+ (86Rb+) release. Radioisotopic methods will be used for these studies. Finally, we will further characterize the induction of lytic activity in cloned CTL by soluble factors. Standard isotope release and microscopic techniques will be used to examine the relative changes in binding and postbinding events as a result of the induction process.